Histones, the nucleosomal proteins that provide the structural framework for compacting the two to four meters of DNA into 90-180 mm of 30 nm diameter chromatin fibers in a typical mammalian cell, are now also known to provide functionalities to the chromatin. We seek to elucidate some of these chromosomal functions of histones. The nucleosome is a complex of about 145 bp DNA and eight histone proteins, two from each of four core histone protein families, H4, H3, H2B and H2A. There is at least another 20 bp of DNA stretching between adjacent nucleosomes which may be complexed with the linker histone H1. Indications that histones may provide more than just a static framework for DNA compaction came from our original discovery in mammals that the H2A histone family subsumes three subfamilies whose members contain characteristic sequence elements that have been conserved independently throughout eukaryotic evolution. Our current interest centers on our demonstration that histone H2AX, one of the three H2A subfamilies, is extensively phosphorylated on a unique serine residue within minutes of the introduction of DNA double-strand breaks into cells, with hundreds to several thousand H2AX molecules becoming phosphorylated per DNA double-strand break in mammals. We named this modified H2AX form g-H2AX. An antibody prepared against the unique g-H2AX epitope showed that g-H2AX is present in discrete foci inside nuclei within minutes of irradiation. The number of foci is consistent with the expected number of DNA double-strand breaks, indicating that g-H2AX forms en masse at the sites of DNA double-strand breaks. The response to ionizing radiation is conserved throughout eukaryotic evolution, occurring not only in mammalian species but also in the non-mammalian species, X. laevis, D. melanogaster, and S. cerevisiae. Studies during the current FY have shown that various proteins involved in DNA repair (Rad50, Rad51, Nbs1, and Brca1) form foci at various times after DNA double-strand break formation at sites of preexisting g-H2AX foci. When g-H2AX foci formation is prevented by preincubation of cells with wortmannin just before irradiation, these DNA repair proteins fail to form foci. Thus there appears to be an ordered recruitment of proteins to sites of DNA damage marked by g-H2AX. Studies this FY in collaboration with other laboratories have also demonstrated the involvement of g-H2AX formation during homologous recombination during meiosis in the mouse and during V(D)J end-joining.